Development and verification of real-time PCR assay for identification of viral agents causing acute respiratory infections in human beings.

ABSTRACT

A multiplex polymerase chain reaction (PCR) for identification of four viruses causing acute respiratory diseases in human beings was developed. The analytical sensitivity of developed RT-PCR for identification of adenovirus, respiratory-syncytial virus, flu viruses types A and B, and actual subtypes of type A flu virus (seasonal and pandemic variants H1N1, seasonal H3N2, and viruses of bird flu that are pathogenic to human beings H5 and H7) was 1 × 103 genome equivalents per milliliter. Diagnostic sensitivity for flu virus type A and B, and also subtypes H1 (seasonal H1N1, pandemic variant of H1N1 of year 2009), H3, H5 was 1 × 103-104 viral particles per milliliter. The method developed has high specificity and does not have positive signal in experiments with DNA/cDNA of human beings and viral DNA. We have studied 50 samples using the developed set. Etiology was defined in 33 samples.